The Golgi complex is the central sorting station for nearly a third of all proteins in eukaryotic cells, but how cells regulate the flow of material through this organelle remains unknown. Protein and membrane traffic into and out of the Golgi is controlled by Rab GTPases that function by recruiting effectors to generate, transport, and tether transport vesicles. The master regulators of these essential Rab pathways are the Rab-GEF proteins that must decide whether to activate their substrate Rabs. The TRAPP complexes are GEFs that activate Rabs at the Golgi, but it is unknown how the timing and magnitude of TRAPP complex activity is regulated. In the budding yeast model, there are two essential Rabs that control Golgi traffic: Ypt1 (Rab1), and Ypt31/32 (Rab11). The TRAPPI complex is the established activator of Ypt1 at early Golgi compartments. The GEF for Ypt31/32 is unknown, and is the subject of controversy. The TRAPPII complex has been proposed to serve as the GEF for Ypt31/32, although firm evidence for this hypothesis is lacking. The uncertainty surrounding the GEF for this critical trafficking pathway is hindering progress in the field. Using a biochemical approach, we have now obtained data that will resolve this controversy. By purifying the TRAPPII complex and performing physiological Rab activation assays, we have discovered that TRAPPII functions as a bone fide GEF for both Ypt1 and Ypt31/32. Importantly, robust GEF activity for Ypt31/32 is strictly dependent upon the presence of membranes, thus explaining why it was not observed previously by other groups, and implying the existence of an autoinhibitory mechanism. Our long-term goal is to uncover mechanisms governing the regulation of Golgi function. The goal of this project is to determine the mechanisms regulating the activation of Ypt1 and Ypt31/32 by the TRAPP complexes. In order to achieve our research goals, we propose the following three aims: 1) Dissect the mechanistic basis for TRAPPII activation of two different Rab GTPases. In vitro assays will be used to investigate how TRAPPII can activate two different substrates, and to ask why membranes are required for activation of Ypt31/32 but not Ypt1. We will then use yeast genetics and cell biology to characterize the functional consequences of perturbing TRAPPII activity in vivo, in order to understand how the TRAPPII complex is regulated in the context of Golgi trafficking. 2) Determine the mechanism and functional significance of TRAPPIII function at the Golgi. Emerging evidence points to TRAPPIII as the physiological GEF for Ypt1 at the early Golgi. We will investigate how TRAPPIII GEF activity is regulated and what role it plays in the cell. 3) Characterize the dynamics of TRAPPII and TRAPIII association with the Golgi. We will determine whether known interacting partners directly regulate the activity and dynamics of TRAPP complexes. We will also determine whether the TRAPP complexes are regulated by crosstalk with other Golgi-localized GTPases. In sum, these studies will lead to a comprehensive model for the regulation of the essential Golgi Rab trafficking pathways.